Breast cancer development is regulated by extracellular biophysical and biochemical cues. Among signaling mediators, members of the Rho family of small GTPases have been identified as controlling the growth, motility, invasion, and metastasis of breast cancer cells. We have previously implicated the splice isoform Rac1b as a key player in activation of epithelial-mesenchymal transition (EMT) and cellular invasiveness in breast cancer cells. Our recently published and preliminary data demonstrate that the key step in Rac1b control of cellular phenotype is through direct interaction with and activation of NADPH oxidase and consequent production of reactive oxygen species (ROS), and that the assembly of the Rac1b-NADPH oxidase complex is controlled by the extracellular matrix (ECM) and tissue tension. We hypothesize that Rac1b acts as a key signaling nexus to integrate mechanical and chemical signaling inputs from the surrounding ECM and tissue to control development of the malignant phenotype. We propose to test this hypothesis through the use of micropatterning, molecular biology, and numerical modeling with cultured cells, transgenic animals, and human breast tissue biopsies. In Specific Aim 1, we will combine engineered substrata with molecular biology approaches to define the molecular mechanisms through which the substratum microenvironment promotes Rac1b membrane localization, NADPH oxidase assembly, and ROS-mediated EMT. We will focus specifically on signaling via integrin-linked kinase (ILK). In Specific Aim 2, we will combine sophisticated three-dimensional engineered and organotypic culture models with experiments using transgenic mice to define how the microenvironment of the normal host epithelium affects Rac1b membrane localization, integrin signaling, EMT, and motility of the resident tumor cells. In Specific Aim 3, we will use breast tissue samples from women who have been found to have benign breast disease to define how age-associated changes in lobular structure and composition affect ILK, Rac1b signaling, and breast cancer risk. The proposed work will significantly advance understanding of how biochemical and biomechanical signals are integrated in the control of Rac1b- and ROS- associated tumor progression and how activation of EMT is controlled by microenvironmental signals. These advances will have direct relevance to our understanding of normal tissue development and progression to malignancy.